The present invention relates to the quantitative determination of the amount of lithium in a liquid test sample. In particular, the present invention is directed to nephelometric and turbidimetric methods for the quantitative measurement of lithium at endogeneous and therapeutic concentrations in biological fluids.
The use of lithium as a mode of treating individuals suffering from acute mania is well known and, accordingly, lithium therapy has become widely accepted as one of the major regimens for the treatment of this condition and for the prevention of relapse in recurrent bipolar mood disorders. In particular, depending upon the severity, the initial dosage of lithium is usually from between about 1,800-3,600 mg/day during acute stages of the condition and 600-1,200 mg/day for less severe, chronic conditions. From between about 1.0-1.6 mequiv./liter during acute therapy and 0.5-1.0 mequiv./liter during chronic therapy are generally considered optimal levels. However, since in some instances indications of toxicity have been observed with blood levels of 1.5 mequiv./liter, and blood levels of 5.0 mequiv./liter are generally considered potentially lethal, close monitoring of blood levels and dose adjustment until an adequate response is achieved is therefore required. Accordingly, there is a need for a convenient, quantitative and standardized clinical procedure, together with corresponding reference ranges, for detecting toxic concentrations of lithium.
Various techniques and methods for the quantitative determination or measurement of lithium in a liquid test medium are known, but, have been limited, for the most part, to flame absorption spectrophotometry and emission flame photometry [Rose, et. al, Am. J. Clin. Pathol., Vol. 74, p. 521(1980)]. These methods require expensive and hazardous instruments and experienced technicians for their operation. Although other methods have been proposed which obviate the need for such instruments, including colorimetric and titrimetric methods, they nevertheless require complicated and often tedious, time consuming procedures to obtain quantitative measurements. Where less tedious or simpler methods have been proposed, quantitation has been sacrificed for semi-quantitative or qualitative results.
In particular, lithium assays and detection protocols have been described which involve a known reaction of iron (Fe.sup.+3) and a salt radical of periodic acid (IO.sub.4.sup.-) with lithium to form a precipitate. The precipitate is then dissolved with a potassium hydroxide solution containing potassium iodate to form a pale yellow precipitate. However, when these reagents are employed in such assays, it is necessary to compare the reaction solution with a sodium chloride test solution when a considerable amount of sodium is present in the test sample. [Procke, et al., Mikrochim. acta, Vol. 3, p. 105 (1938)].
The gravimetric analysis of lithium in a prepared test solution where lithium is precipitated as a complex periodate by a strongly alkaline potassium periodate solution has also been described. The lithium periodate precipitate is then removed by filtration, dissolved in dilute sulfuric acid, and the periodate titrated with a standardized thiosulfate or arsenite solution. [Rogers, et al., Ind. Eng. Chem., Anal. Ed., Vol. 15, p. 209 (1943)]. Although lithium can be distinguished from other alkali metals, all metals except those of the alkali group must first be removed according to such method, as well as ammonia when more than a few milligrams are present.
In a volumetric method for the determination of lithium, the quantitative precipitation of lithium fluoride in alcohol for the analysis of concentrated solutions of lithium chloride and lithium nitrate is described. [Baumann, Analytical Chemistry, Vol. 40, No. 11, p. 1731 (1968)]. However, this technique lacks sensitivity and selectivity over sodium and, moreover, since it is performed in an alcohol solution, it could not be used for analyzing serum samples without prior removal of endogenous protein.
A colorimetric method for the semi-quantitative determination of lithium in serum pretreated with trichloroacetic acid has also been described [Plum, Clinica Chimica Acta, Vol. 2, p. 67(1957)]. According to such method, the pretreated serum is combined with potassium hydroxide, sodium chloride, and a reagent comprising potassium metaperiodate, potassium hydroxide and ferric chloride. The resulting reaction solution containing a yellow lithium precipitate was then spectrophotometrically analyzed at 720 nm., and an attempt to construct a standard curve (optical density vs. time) employing an end-point analysis was made. However, such attempt was unsuccessful due to the presence of lipids in the test sample and, accordingly, such method does not provide an exact or quantitative determination of lithium.
In particular, such method involves tedious sample pretreatment steps which remove the endogenous turbidity associated with varying serum proteins, but which do not remove lipids. Since it has been shown that the presence of lipids contributes to the background optical density of a test sample, any variation in serum lipid content, even after removal of the serum proteins, could cause variability in the apparent lithium content according to such method. Furthermore, since it is known that the time of the maximum optical density change per second decreases as the lithium concentration increases, a sample blank from the optical density values would still be unsuccessful. Concomittantly, the absolute optical density of a sample of high lithium content is proportionately higher than that of a sample of low lithium content for early read times and proportionately lower than a sample of low lithium content for later read times. Therefore, taking an optical density measurement at a fixed time would yield a nonlinear standard curve, the shape of which would be determined by the exact read time. While lack of linearity does not preclude quantitation, the optical density of the sample blank does not change with time. Subtraction of a sample blank would therefore have different effects on samples of different lithium content. Therefore lipids would nevertheless still interfere with the lithium determination with sample blanking. In particular, quantitation cannot be achieved by reading optical density at a fixed read time with or without sample blanking, because if the sample incubation is permitted to continue for periods of time even greater than 300 seconds, the particles responsible for the turbidity aggregate and drop out of solution to cause considerable and untolerable variability.
A method employing similar reagents has also been described, but also provides only qualitative results. [Feigl, Spot Tests In Inorganic Analysis, Elsevier Publishing Co., New York, N.Y., 5th ed., p. 233 (1958)].
Accordingly, it is an object of the present invention to provide a quantitative method for the detection of therapeutic concentrations of lithium in a liquid test sample, particularly biological test samples.
Another object of the present invention is to provide a quantitative method for the detection of lithium in a liquid test sample employing instruments which are safe, convenient, and easily operated.
Further, it is an object of the present invention to provide a quantitative method for the detection of lithium in a liquid test sample which does not require the removal or minimization of lipids or proteins present in a serum sample.